The effects of ions on the conjugation of xenobiotics by the aralkyl-CoA and arylacetyl-CoA N-acyltransferases from bovine liver mitochondria.
The aralkyl-CoA:glycine N-acyltransferase and the arylacetyl-CoA:amino acid of N-acyltransferase were purified from bovine liver mitochondria and their response to a variety of ions investigated. The activity of the aralkyl transferase was inhibited by divalent cations with all substrates investigated. For benzoyl-coenzyme A (CoA), K+ was a competitive inhibitor, competing for binding at the benzoyl-CoA binding site. With salicylyl-CoA, K+ did increase the dissociation constant (KD) for acyl-CoA but it was not a competitive inhibitor and in addition, K+ increased the Michaelis constant for glycine (Kglym) tenfold. The data suggest that the increase in Kglym is due to bound K+ forcing reorientation of salicylyl-CoA at the active site so that it impinges on the glycine binding site. Inorganic anions and cations did not affect the extent of product inhibition by hippuric acid with either acyl-CoA and this was because they affected the binding of acyl-CoA and hippuric acid to the same extent. Ions did, however, greatly reduce the extent of product inhibition by CoA. This is critical because under approximate in vivo conditions (2.5 mM CoA), the salt-free enzyme would be almost completely inhibited by CoA. The arylacetyl transferase was activated by inorganic ions when assayed at saturating substrate concentrations. However, at physiologic concentrations of glycine certain salts were modestly inhibitory. The inhibitory effect of KCl was characterized by a large decrease in the affinity of the enzyme for phenylacetyl-CoA, suggesting that the arylacetyl-CoA region of the active site contained an inhibitory ion binding site. At low (physiologic) concentrations of substrate, the arylacetyl transferase was extensively inhibited by CoA and this inhibition was greatly reduced by ions. The 3'-phosphate group on CoA was found to be important for binding to the salt-free enzyme but in the presence of ions its importance was diminished. In the absence of inorganic ions the affinity of the enzyme for phenylacetyl-CoA and naphthylacetyl-CoA was so high that it could not be measured. In the presence of KCl the KD values for phenylacetyl-CoA and naphthylacetyl-CoA were similar, but the Km for glycine was extremely high for 1-naphthylacetyl-CoA conjugation, which accounts for its slow rate of metabolism. Conjugation with glutamine had a high Michaelis constant for glutamine (KGlum) and a low maximum velocity (Vmax) which accounts for the absence of glutamine conjugation in vivo.
Kelley
M
M
Liver Study Unit, Veterans Affairs Medical Center, San Francisco, CA 94121.
Vessey
D A
DA
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism*
D015262:Xenobiotics; D011189:Potassium Chloride; D000217:Acyltransferases; D000123:Acetyltransferases; C067287:arylacetyl-CoA N-acetyltransferase; C020680:glycine acyltransferase